High molecular weight desulphatohirudin

ABSTRACT

PCT No. PCT/EP94/02438 Sec. 371 Date Mar. 29, 1995 Sec. 102(e) Date Mar. 29, 1995 PCT Filed Jul. 23, 1994 PCT Pub. No. WO95/04823 PCT Pub. Date Feb. 16, 1995The invention pertains to the field of thrombin inhibitors and describes the production of modified desulphatohirudins, particularly desulphatohirudins, with the aid of genetic engineering. The present invention is also directed to a method for the denaturation and renaturation of a modified desulphatohirudin to a biologically active thrombin inhibitor and the combination of two or more of desulphatohirudins to form higher molecular weight thrombin inhibitors.

The invention pertains to the field of thrombin inhibitors and describesthe production of modified hirudins more especially desulphatohirudinmuteins, with the aid of genetic engineering. It is a further object ofthe invention to provide a method for the preparation of biologicallyactive high molecular weight hirudin by combining two to four monomersof said hirudins.

The hirudins are anticoagulant agents that occur naturally in leeches(e.g. in medicinal leech Hirudo medicinalis). The hirudins are equallyacting polypeptides having an accumulation of hydrophobic amino acids atthe N-terminus and of polar amino acids at the C-terminus, threedisulfide bridges and the anticoagulant activity in common. Acharacteristic feature of most natural hirudins is the presence of atyrosine sulphate residue at the C-terminal part (Tyr⁶³) of themolecules. Apart from the well-known hirudin variants HV1, HV2 and HV3additional hirudins have been reported to exist in nature, see, forexample, M. Scharf et al. FEBS Lett. 255, 105-110 (1989), supporting theconcept of hirudins as a family of isoinhibitors.

The hirudins, for example hirudin variant 1 (HV1), are the most potentand most specific known inhibitors of thrombin, the serine protease thatcatalyzes the final step (the conversion of the zymogen fibrinogen inclottable fibrin) in blood coagulation. Other enzymes of the bloodcoagulation cascade are not inhibited by hirudins. In contrast toheparin which is the preferred anticoagulant in conventionalanticoagulation therapy, the hirudins exert their inhibiting actiondirectly on thrombin and, unlike the former, do not act throughantithrombin III. The only pharmacologically detectable effect ofpurified hirudins is the inhibition of blood coagulation and theprophylaxis of thrombosis. No side effects, such as effects on heartrate, respiration, blood pressure, thrombocyte count, fibrinogen andhemoglobin, have been observed after intravenous administration ofhirudins to dogs, even in high doses. In a series of animal modelshirudins have proved effective in experimental thrombosis (inducedeither by stasis or by the injection of thrombin), in endotoxin shock,and also in DIC (disseminated intravascular coagulation). Wheneverdirect comparison tests have been carried out, hirudins have proved tobe superior to heparin.

In recent years cDNAs and synthetic genes coding for hirudin variantshave been cloned and expressed in microbial hosts, such as Escherichiacoli and, in particular, Saccharomyces cerevisiae. Although theexpression products lack the sulphate monoester group at Tyr⁶³ - andwere therefore designated "desulphatohirudins"--they turned out toexhibit essentially the same biological activity as the naturalsulphated hirudins.

A characteristic of the therapeutic application of recombinantdesulphatohirudin is its half-life in the circulation of about 50 min.and therefore, the rapid excretion from the human body. The reason forthe rapid excretion is the filtration of the glomerulum in the kidneyfor substances having a molecular weight below 70 000. Because of thisrapid excretion, the daily dose of desulphatohirudin is administerednormally in two or more separate portions. One strategy to obtain longeracting thrombin inhibitors is to synthesize high molecular weighthirudins. In WO 91/08229 the conjugation of hirudin withpolyalkylenglycol is described. As the polyalkylenglycols are normallyvery heterogeneous in molecular size and weight, their combination withhirudin leads to a heterogeneous mixture and the administration of adefined dose of hirudin is difficult. In another approach the hirudin iscrosslinked with other proteins like albumin (WO 92/05748). These kindsof conjugates show diminished activities and increased immunogenicproperties and are therefore unsuitable for long term administration.

In WO 91/09125 relatively inactive fusion proteins are described thathave to be activated by enzymes of the clotting cascade to havefibrinolytic or clot formation inhibition activity.

Surprisingly it has now been found that conjugates consisting of two tofour residues of desulphatohirudin muteins or desulphatohirudinderivatives have a significantly increased plasma half life even if themolecular weight is considerably lower than the critical value of about70 000, see above, surprisingly show no detectable increased immunogenicproperties, do not have to be cleaved by an enzyme involved in bloodclotting and have an unchanged activity. With this increased plasma halflife the administration of the daily dose of desulphatohirudin in asingle portion is possible.

Accordingly, the present invention relates to a conjugate essentiallyconsisting of two to four residues of desulphatohirudin muteins orderivatives wherein the conjugate is no fusion protein and the residuesof desulphatohirudin muteins or derivatives having hirudin activity arenot connected via glutaraldehyde or carbodiimide.

The term "desulphatohirudin" is intended to embrace alldesulphatohirudin compounds described in literature or obtainable from atransformed microorganism strain containing DNA which codes for adesulphatohirudin or a derivative thereof. Such desulphatohirudins are,for example, desulphatohirudin derivatives HV 1, HV2 and HV3 (PA), aswell as other hirudin and hirullin proteins as described by M. Scharf etal. (FEBS Lett: (1989), 255, 105-110) and EP-A-347 376. It is to beunderstood that hirudin derivatives or shorter fragments having hirudinactivity (i.e. having a thrombin inhibiting and/or thrombin bindingaction) are also covered by the term "desulphatohirudin". Such fragmentsand derivatives are, for example, C-terminally shorteneddesulphatohirudins, i.e. desulphatohirudins lacking up to seven aminoacids at the C-terminus.

The desulphatohirudins or active fragments or derivatives thereof can bemodified by a linkage of a further reactive group, wherein the reactivegroup shows a biological effect on the thrombin activity. It is forexample possible to combine the thrombin binding domain ofdesulphatohirudin that is located in the C-terminal part with achemically synthesized thrombin inhibitor. Examples for such chemicallymodified desulphatohirudin derivatives are hirulogs (WO 90/04642).

In a preferred embodiment a conjugate according to the inventionessentially consists of two residues of desulphatohirudin muteins orderivatives. The components of the conjugates according to theinvention, i.e. the residues of desulphatohirudin muteins orderivatives, may be the same or different.

The desulphatohirudin muteins or derivatives are connected directly orvia a linker group. For this purpose normally one or more, preferred isone, genuine amino acid of desulphatohirudin is replaced by one or moreradicals capable of being crosslinked, e.g., an amino acid bearing areactive side chain.

It is an object of the invention to provide a desulphatohirudin muteinor derivative capable of building conjugates, essentially conjugatesconsisting of two to four residues of such desulphatohirudins. In apreferred embodiment of the invention one or more genuine amino acids ofa desulphatohirudin mutein or derivative are replaced by an amino acidselected from the group consisting of aspartic acid, glutamic acid,lysine and cysteine.

The replacement of one amino acid is preferred, more preferred is thereplacement of a single amino acid by cysteine and most preferred thereplacement of Asp³³ in desulphatohirudin HV1 by cysteine.

A conjugate consisting of two desulphatohirudin residues can be producedby forming a peptide bond between aspartic acid or glutamic acid in oneresidue and lysine in the other residue, or preferred a disulfide bondbetween two cysteine residues.

In the alternative, the components of the conjugate according to theinvention (two to four residues of desulphatohirudin mutein ordesulphatohirudin derivatives) may also be connected via a linker group.To this end, desulphatohirudin muteins or derivatives are linked via thereactive side chain, e.g., of the introduced amino acid as defined aboveto a linker molecule which has two or more reactive groups, two reactivegroups being preferred, said reactive groups being capable of reactingand forming a covalent bond with the reactive side chain of thedesulphatohirudin mutein or derivative. Such linker molecules aremolecules beating two or more reactive groups like --SH, --N₃, --COOH,--COBr, --COCl, --NH₂, --CHO, --CO--O--CO--, --CO--NH--CO--. Examplesare N-5-azido-2-nitrobenzoyloxysuccinimide, p-azidophenazylbromide,p-azidophenyl glyoxal, N-4-(azidophenylthio)phthalimide,bis(sulfosuccinimidyl) suberate, bis-maleimidohexane,bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone,1,5-difluoro-2,4-dinitrobenzene, 4,4'-diisothiocyano-2,2'-disulfonicacid stilbene, dimethyl adipimidate, dimethyl pimelimidate, dimethylsuberimidate, dithiobis(succinimidylpropionate), disuccinimidylsuberate, disuccinimidyl tatrate, dimethyl 3,3'-dithiobispropionimidate,4,4'-dithiobisphenylazide, 3,3'-dithiobis(succinimidylpropionate),ethyl-4-azidophenyl-1,4-dithiobutyrimidate,1-azido-4-fluoro-3-nitrobenzene, N-hydroxysuccinimidyl-4-azidobenzoate,methyl-4-azidobenzoimidate,m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester,N-hydroxysuccinimidyl-4-azidosalicylic acid,p-nitrophenyl-2-diazo-3,3,3-trifluoro propionate,N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate, sulfosuccinimidyl2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate,N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate,sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-di-thiopropionate,N-succinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, succinimidyl4-(p-meleimidophenyl)-butyrate, N-succinimidyl3-(2-pyridyldithio)propionate,bis[2-(sulfosuccinimidooxy-carbonyloxy)ethyl]sulfone,disulfosuccinimidyl tatrate, ethyleneglycolbis(sulfosuccinimidylsuccinate),m-maleimidobenzoyl-N-hydroxysulfosuccinate), sulfosuccinimidyl(4-azidophenyldithio)-propionate, sulfosuccinimidyl6-(4'azido-2'-nitrophenylamino)hexanoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl4-(p-maleimidophenyl)butyrate and 2-iminothiolane.

Preferred linker molecules specifically react with --SH groups of aminoacids, e.g., dithio compounds like dithioerythrol, dithiothreitol,dimercaptobenzene, 1,4-butandithiole, 1,3-propanedithiol, short aminoacid chains beating two cysteins or other --SH crosslinking agents likebis-maleimidohexane, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, succinimidyl4-(p-meleimidophenyl)-butyrate, N-succimimidyl3-(2-pyridyldithio)propionate,m-maleimidobenzoyl-N-hydroxysulfosuccinyte).

The amino acid exchange and therefore, the attachment to anotherdesulphatohirudin or to a linker is done in a region not essential forthe activity of desulphatohirudin. Desulphatohirudin contains twofunctional domains which bind to independent sites of α-thrombin; acompact N-terminal core domain binding to the catalytic site of thrombin(Chang et al., J. Biol. Chem. (1990), 265, 22159-22166) and a disorderedterminal tail which is complementary to the exosite (fibrinogenrecognition site) of the enzyme Maraganore et at., J. Biol. Chem.(1989), 264, 8692-8698). Linking two monomers through the C-terminalregion is not desirable as nearly every amino acid residue inside thisdomain is required for the full potency of desulphatohirudin (Chang, J.Biol. Chem. (1990), 265, 22159-22166). Within the N-terminal domain,however, there is a flexible loop (residues 30 to 40, especiallyresidues 31 to 36) which is located on the surface and is distant fromthe thrombin binding site (Grutter et al., EMBO (1990), 9, 2361-2365).The alteration of the desulphatohirudin monomers and the connection ofthe desulphatohirudin monomers to form the conjugates according to theinvention is preferably done in the region between amino acid residues 7to 50, more preferably 30 to 40 and especially between residues 31 to36. Most preferred is a desulphatohirudin HV1 wherein Asp³³ is replacedby cysteine. Therefore the monomers of the desulphatohirudin muteins orderivatives, or the monomer of desulphatohirudin mutein or derivativeand the linker group, are preferably linked in the region between aminoacid residues 7 to 50, more preferably 30 to 40, and most preferablybetween residues 31 to 36.

Due to the amino acid exchange, the folding of the nascent modifieddesulphatohirudin chain may be different to the folding of theunmodified desulphatohirudin. If the native desulphatohirudin structureis not formed spontaneously, the isolated desulphatohirudin may bedenaturated and refolded under suitable conditions to a configurationthat shows thrombin inhibiting activities.

A number of publications have appeared which report refolding attemptsfor individual proteins produced in bacterial hosts, or which areotherwise in a denatured or non-native form. Formation of a dimeric,biologically active human colony stimulating factor-1 (CSF-1) afterexpression in E. coli is described in WO 88/8003 and by Halenbeck, R. etal. Biotechnologie (1989), 7, 710-715. The procedures described involvethe steps of initial solubilization of CSF-1 monomers isolated frominclusion bodies under reducing conditions in a chaotropic environmentcomprising urea or guanidine hydrochloride, refolding which is achievedby stepwise dilution of the chaotropic agents, and final oxidation ofthe refolded molecule in the presence of a redox-system. In WO 88/8849 aprocess for recovering recombinant interleukin-2 (IL-2) is disclosed,characterized in that IL-2 isolated from refractile bodies is denaturedunder reducing conditions with 6M guanidine hydrochloride, the solubleIL-2 is oxidized by a controlled oxidation in the presence of Cu²⁺ ions,and the oxidized IL-2 refolded by reducing the concentration of thedenaturant in the solution. Interleukin-2 and interferon-β have beenrefolded using SDS for solubilization and Cu²⁺ ions as oxidationpromoters of the fully reduced protein (U.S. Pat. No. 4,572,798). Theprocess for isolating recombinant refractile proteins as described inU.S. Pat. No. 4,620,948 involves strong denaturing agents to solubilizethe proteins, reducing conditions to facilitate correct folding anddenaturant replacement in presence of air or other oxidizing agents toreform the disulfide bonds. A method for renaturing unfolded proteinsincluding cytochrome c, ovalbumin and trypsin inhibitor by reversiblybinding of the denatured protein to a solid matrix and stepwiserenaturing it by diluting the denaturant is disclosed in WO 86/5809. Theforegoing references are merely representative of a huge amount ofliterature dealing with the refolding of non-native proteins derivedfrom different sources. The man skilled in the art on the other handknows that the success of refolding experiments cannot be predicted.Unsucessful experiments are usually not reported. There is no certaintythat anyone of the reported refolding conditions would work at all witha given denatured protein such as desulphatohirudin, muteins orderivatives. Considering the fact, that unmodified desulphatohirudincontains 6 cysteine residues and desulphatohirudin HV1 wherein Asp³³ isreplaced by cysteine contains 7 cysteine residues per chain and a numberof intramolecular disulfide bonds, which are required for activity, itis a particularly difficult challenge to produce biologically activepolymeric and especially dimeric desulphatohirudin from its monomeric,denatured or otherwise non-native form.

It is therefore an object of the invention to provide a method forunfolding and refolding desulphatohirudin muteins or derivatives to abiologically active polymeric and especially dimeric form comprising thesteps:

denaturing of desulphatohirudin as defined above with chaotropic agent,

renaturing of desulphatohirudin under refolding conditions, and, ifrequired

linking of the desulphatohirudins by means of a linker, or directly.

Chaotropic agents which in suitable concentration are capable ofeffectively denaturing proteins by changing the spatial configuration ofthe respective protein through alterations at the surface thereof,either through altering the state of hydration, the solvent environment,or the solvent-surface interaction in aqueous solution and are wellknown in the art. Examples of such chaotropic agents or denaturantsinclude urea, guanidine hydrochloride, sodium thiocyanate atconcentrations in the range of about 4 to 9M, and detergents such asSDS, which are supplied in concentrations in the order of 0.01 to 2%.Also, acidification of the aqueous solution to a pH below 4 or basicconditions of a pH above 10 and elevated temperatures will result indenaturation of the monomer.

The term "refolding conditions" refers to buffer conditions wherein thedenatured monomer is permitted to assume a conformation associated withbiological activity. Conventional buffer systems such as Tris,phosphate, carbonate or citrate buffers can be used at a pH range ofabout 6 to 10. Under refolding conditions disulfide bond formation ispromoted. Such conditions include, e.g., the presence of a solubilizingagent and a redox system which permits the continuous oxidation andreduction of the thiol/disulfide pairs. The buffer system mayadditionally contain suitable salts.

Suitable solubilizing agents are detergents, preferably mild detergents,organic, water-miscible solvents, or phospholipids or mixtures thereof.

Suitable redox systems which encourage the formation of disulfides are,e.g., low molecular weight sulfhydryl/disulfide reagent combinationssuch as glutathione in its oxidized and reduced form, dithiotreitol inits oxidized and reduced form, β-mercaptoethanol or β-mercaptomethanolin its oxidized and reduced form, cysteine and its reduced form, andcystamine and its reduced form at concentrations of about 1 to 100 mM,especially of about 1 to 10 mM. Alternatively, thioredoxin ordisulfideisomerases at a concentration of about 10 to 1000 μg/ml,especially 50 to 200 μg/ml can be used instead of the low molecularweight sulfhydryl/disulfide system.

Optionally, to further promote intermolecular and intramoleculardisulfide formation, an effective amount of an oxidation promoting agentcontaining Cu²⁺ ions (such as CuCl₂, Cu(NO₃)₂ or o-phenanthroline/Cu²⁺complexes) or Fe³⁺ ions (such as FeCl₃ or Fe₂ (SO₄)₃) might be added tothe refolding buffer. An effective amount is the amount which at minimumwill be necessary to conduct the oxidation of sulfhydryl groups within aconvenient time period and which is approximately equivalent to theconcentration of free sulfhydryl groups in the desulphatohirudin muteinsor derivatives, which are destined to be involved in forming the desireddisulfide bonds. Preferable mounts range between 0.001 to 100 μM.

Furthermore, O₂ or air may be bubbled through the refolding buffereither in presence or absence of oxidation promoting agents. Oxidationmay also be performed using I₂ or benzoquinone derivatives.

In the case of desulphatohirudin variant 1 wherein, e.g., Asp³³ isreplaced by cysteine ([Cys³³ ]HV1) the denaturation, folding anddimerization steps are usually carried out after isolation of thedesulphatohirudin by methods known per se to obtain a homogenousproduct. The initial isolated desulphatohirudin is, e.g., denatured byaddition of guanidine hydrochloride under reducing conditions, refoldedby removal of the guanidine hydrochloride under alkaline conditions andsubsequently the [Cys³³ ]HV1 monomers are oxidized and simultaneouslydimerized to the [Cys³³ ]HV1--[Cys³³ ]HV1 dimers.

Both the Cys³³ monomer and the dimer are highly active anticoagulantsand their potencies of thrombin inhibition are nearly indistinguishable(±3%). This suggests that one desulphatohirudin dimer binds to twomolecules of α-thrombin.

In the case of a synthetic linker the monomers of the refoldeddesulphatohirudin muteins or derivatives can be attached to the linkerby manners known pre se. Reactive amino acids that should not bind tothe linker can be protected by usual protective groups as described forexample in "Protective Groups in Organic Chemistry", Plenum Press,London, New York 1973, in "Methoden der organischen Chemie",Houben-Weyl, 4th Edition, Vol. 15/1, Georg-Thieme-Verlag, Stuttgart1974, and in Th. W. Greene, "Protective Groups in Organic Synthesis",John Wiley & Sons, New York 1981. It is characteristic of protectinggroups that they can be readily removed, that is to say withoutundesired secondary reactions taking place, for example by solvolysis,reduction or photolysis.

The alterations in the amino acid sequence of desulphatohirudin muteinor derivatives can be made by site directed mutagenesis, if theintroduction of genetically encoded amino acids is desired. Otherwise,at least the part of desulphatohirudin mutein or derivative bearing thealteration has to be synthesized chemically, e.g., by fixing a firstgenetically synthesized part of the desulphatohirudin mutein orderivative to a solid phase and adding the missing radicals to the fixedpolypeptide, in a manner known per se.

For convenience, modification of desulphatohirudin, i.e., alteration ofan amino acid, is not done at the protein level. Instead, it isadvantageous to modify the gene coding for desulphatohirudin by sitedirected mutagenesis in such a way that upon expression of said modifiedgene by a host the desired desulphatohirudin mutein is produced in whichone or more of the amino acids is (are) replaced by one or more aminoacids capable of forming bonds to a further desulphatohirudin or alinker. In a preferred embodiment a single amino acid is replaced bycysteine.

The method for the production of desulphatohirudin mutein according tothe invention comprises culturing under appropriate nutrient conditionstransformed host cells containing a DNA sequence coding for a hirudinmutein in which one or more of the amino acids is (are) replaced by oneor more amino acids capable of forming bonds to a furtherdesulphatohirudin or a linker, and isolating said desulphatohirudinmutein.

Suitable host cells are, e.g., bacteria, such as E. coli, or eukaryotichost cells, e.g., yeast, such as Saccharomyces cerevisiae. The preferredhost organism according to the present invention is Saccharomycescerevisiae.

The host cells, especially yeast, containing the above DNA sequence arecultured using methods known in the art.

The DNAs including the sequences as shown in SEQ ID NO: 5 and 7 can bemanufactured by methods known in the art. The methods for themanufacture of these DNA include chemically synthesizing the DNA,excising a portion of the DNA comprising the codon for the undesiredamino acid residue from the genuine hirudin gene and replacing it with aDNA segment wherein said codon has been substituted with adeoxyribonucleotide triplet coding for the desired amino acid residue,or accomplishing the deoxyribonucleotide substitution by means ofsite-directed mutagenesis.

The chemical synthesis of DNA is well-known in the art and makes use ofconventional techniques. Appropriate techniques have been compiled by S.A. Narang (Tetrahedron (1983), 39, 3). In particular, the methods aredescribed in EP-A-146 785.

Excision of a portion of the mature hirudin DNA may be effected by usingrestriction enzymes. A prerequisite of this method is the availabilityof appropriate restriction sites in the vicinity of the codon to bealtered. A small restriction fragment containing the codon for anundesired amino acid is removed by endonuclease cleavage. Acorresponding double stranded DNA sequence is prepared, for example bymeans of chemical synthesis, in which triplets coding for the desiredamino acid are used. The DNA fragment is ligated in the properorientation to the remaining large fragment to yield a double strandedDNA sequence coding for a desulphatohirudin mutein. For convenience andin order to facilitate handling of the hirudin gene the latter isadvantageously contained in a greater DNA segment provided withappropriate linkers which allow insertion and cloning of the segment ina cloning vector.

In a preferred embodiment of the present invention the preparation ofDNAs having the DNA sequences according to SEQ ID NO: 7 is effected bysite-directed mutagenesis. This method is an in vitro mutagenesisprocedure by which a defined site within a region of cloned DNA can bealtered (cf. the review articles of M. J. Zoller and M. Smith, MethodsEnzymol. (1983), 100, 468; D. Botstein and D. Shortle, Science (1985),229, 1193).

The method of mutating the wild-type hirudin gene is characterized inthat the single-stranded wild-type hirudin gene or a single-stranded DNAcomprising the wild-type hirudin gene is hybridized to anoligodeoxyribonucleotide primer which is complementary to the region ofthe hirudin gene to be mutated except for mismatch(es) that direct(s)the mutation, the hybridized oligodeoxyribonucleotide is used as aprimer to initiate the synthesis of the complementary DNA strand, e.g.via PCR, the resulting (partially) double-stranded DNA is transformedinto a recipient microorganism strain, the microorganism strain iscultivated and transformants containing DNA with the modified hirudingene are selected.

In a preferred form two oligodeoxynucleotide primers (1) and (4) areused that are complementary to nucleotide sequences upstream anddownstream of the desired sequence for amplification of the templateDNA; and two internal primers (2) and (3) are used that are partlycomplementary to each other and both cover the site of mutation. The PCRprimers (1) and (3) and the primers (2) and (4) are used pairwise inseparate reactions to amplify parts of the template DNA and to introducethe mutation. In a second reaction both products are mixed and amplifiedwith primers (1) and (4).

The wild-type hirudin gene is preferably contained in a plasmid, forexample a plasmid described in EP-A-143 081 or EP-A-340 170. Formutagenesis the hirudin gene is advantageously isolated from theplasmid, for example by restriction endonuclease digestion resulting ina double-stranded DNA comprising the hirudin gene and optionally otherfunctions linked thereto, for example promoter, signal peptide DNA etc.,and having sticky ends, and amplified via PCR or ligated to a suitablerestriction fragment of the replicative form (RF) of a single-strandedDNA phage, such as a derivative of phage M13, e.g., M13mp8 or M13mp9.Competent microorganisms, e.g., Ca-treated cells of an E. coli strain,are transfected with the hybrid phage vector and single-stranded phageDNA containing the inserted hirudin gene is isolated from thesupernatant of the cultured cells. To this template the mismatch(mutagenic) oligodeoxyribonucleotide primer is hybridized.

Some of the methods described in the literature depend on the enzymaticextension of the mutagenic primer along the circular template followedby ligation to produce a covalently closed double-stranded circularmolecule having the mutation(s) in the newly synthesized strand. Due tothe generally low efficiency of this process a modified procedure hasbeen developed (K. Norris et al., Nucl. Acids Res. (1983), 11, 5103) inwhich a second primer is used. In the case of an M13 phage vector auniversal M13 sequencing primer is preferably used (EP-A-225 286).

Hybrid vectors containing DNA sequences coding for desulphatohirudinmuteins

The invention relates also to hybrid vectors comprising a promoter and aDNA sequence coding for a human desulphatohirudin mutein in which one ormore of the amino acids is (are) replaced by one or more amino acidscapable of forming bonds to a further desulphatohirudin or a linker,which DNA sequence is under transcriptional control of said promoter.

The hybrid vectors according to the invention are selected from thegroup consisting of a hybrid plasmid and a linear DNA vector and arefurther selected depending on the host organism envisaged fortransformation.

Promoters for yeast which are the most preferred host organismsaccording to the present invention are derived from the genomic DNA ofyeast, especially of Saccharomyces cerevisiae. Preferably, the promoterof a highly expressed yeast gene is used for the expression ofdesulphatohirudin mutants. Thus, the promoter of the TRP1 gene, the ADHIor ADHII gene, acid phosphatase (PHO5) gene, CUP1 gene, isocytochrome cgene, or a promoter of the genes coding for glycolytic enzymes, such asTDH3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a shortenedversion of GAPDH (GAPFL) which is preferred, 3-phosphoglycerate kinase(PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, invertaseand glucokinase genes, or a promoter of the yeast mating pheromone genescoding for the a- or α-factor, can be used. Preferred vectors of thepresent invention contain promoters with transcriptional control.Promoters of this type, e.g. the promoter of the PHO5 or the CUP1 gene,can be turned on or off by variation of the growth conditions. Forexample, the PHO5 promoter can be repressed or derepressed at will,solely by increasing or decreasing the concentration of inorganicphosphate in the medium. Thus, the promoter can be repressed during theexponential growth phase of the yeast and may be turned on (derepressed)only during early stationary phase at maximal cell density allowingexpression of the gene controlled by the PHO5 promoter. The CUP1promoter can be turned on by the addition of Cu²⁺ -ions to the medium,e.g., in the form of a copper salt.

In the hybrid vectors of the present invention, the promoter is operablylinked to the mutant hirudin coding region so as to ensure effectiveexpression of the desulphatohirudin mutein. In the preferred embodimentof the present invention, especially if yeast is used as the hostorganism, a signal sequence is included in the construction. Suitablesignal sequences are those of secreted yeast gene products, e.g., thePHO5, invertase or α-factor signal sequences, or the hirudin signalsequence. Alternatively, fused signal sequences may be constructed by inframe ligating part of the signal sequence of the gene naturally linkedto a yeast promoter, with part of the hirudin signal sequence. Thosecombinations are favored which allow a precise cleavage between thesignal peptide and the mature hirudin amino acid sequence. Additionalsequences, such as pro-or spacer-sequences which may or may not carryspecific processing signals can also be included in the constructions tofacilitate accurate processing of precursor molecules. Alternativelyfused proteins can be generated containing internal processing signalswhich allow proper maturation in vivo or in vitro. Preferably, theprocessing signals contain a Lys-Arg residue, which is recognized by ayeast endopeptidase located in the Golgi membranes. Upon expression ofthe mutant hirudin gene, the gene product enters the secretory pathwayand is transported to the periplasmic space. If further excretionthrough the cell wall into the culture broth can be achieved, aconsiderable increase in yields should be possible. Also the recoveryprocess can be simplified with no cells to be disrupted.

Preferably, the hybrid vectors according to the present inventioncomprise also the 3'-flanking sequence of a gene which contains theproper signals for transcription termination and polyadenylation.Suitable 3'-flanking sequences are for example those of the genenaturally linked to the promoter used, or 3'-flanking sequences of otheryeast genes, e.g., of the PHO5 gene.

In the preferred hybrid plasmids according to the invention, theadditional DNA sequences carry a yeast replication origin and aselective genetic marker for yeast. Hybrid plasmids containing a yeastreplication origin, e.g. an autonomously replicating segment (Ars), areextrachromosomally maintained within the yeast cell after transformationand are autonomously replicated upon mitosis. Hybrid plasmids containingsequences homologous to yeast 2μ plasmid DNA can be used as well. Thesehybrid plasmids will get integrated by recombination into 2μ plasmidsalready present within the cell or will replicate autonomously. 2μsequences are especially suitable for high-frequency transformationplasmids and give rise to high copy numbers.

In addition, the preferred hybrid plasmids according to the inventionmay include a DNA sequence as part of a gene present in the host yeastchromosome (e.g. the PHO5 gene or its promoter linked to the mutanthirudin coding region). By virtue of the homologous sequence, whichshould amount to a stretch of about 20 to 100 deoxynucleotides, thewhole plasmid or linear fragments thereof can be stably incorporatedinto the host chromosome by recombination. Thus, during propagation theprogeny cells will retain the introduced genetic material even withoutselective pressure.

As to the selective gene marker for yeast, any marker gene can be usedwhich facilitates the selection for transformants due to the phenotypicexpression of the marker. Suitable markers for yeast are particularlythose expressing antibiotic resistance or, in the case of auxotrophicyeast mutants, genes which complement host lesions. Corresponding genesconfer, for example, resistance to the antibiotic cycloheximide orprovide for prototrophy in an auxotrophic yeast mutant, for example theURA3, LEU2, HIS3 or TRP1 gene. It is also possible to employ as markersstructural genes which are associated with an autonomously replicatingsegment providing that the host to be transformed is auxotrophic for theproduct expressed by the marker.

Advantageously, the additional DNA sequences which are present in thehybrid plasmids according to the invention also include a replicationorigin and a selective genetic marker for a bacterial host, especiallyEscherichia coli. There are useful features which are associated withthe presence of an E. coli replication origin and an E. coli marker in ayeast hybrid plasmid. Firstly, large amounts of hybrid plasmid DNA canbe obtained by growth and amplification in E. coli and, secondly, theconstruction of hybrid plasmids is conveniently done in E. coli makinguse of the whole repertoire of cloning technology based on E. coli. E.coli plasmids, such as pBR322 and the like, contain both E. colireplication origin and E. coli genetic markers conferring resistance toantibiotics, for example tetracycline and ampicillin, and areadvantageously employed as part of the yeast hybrid vectors.

The additional DNA sequences which contain, for example, replicationorigin and genetic markers for yeast and a bacterial host (see above)are hereinafter referred to as "vector DNA" which together with theyeast promoter and the mutant hirudin coding region is forming a hybridplasmid according to the invention.

The preferred hybrid vectors of the present invention are prepared bymethods known in the art, for example by linking a yeast promoter, amutant hirudin coding region, the 3' flanking sequence of a yeast geneand optionally vector DNA.

For the preparation of hybrid plasmids, conveniently mapped circularvector DNA, for example bacterial plasmid DNA or the like (see above),having at least one restriction site, preferably two or more restrictionsites, can be employed. Advantageously, the vector DNA already containsreplication origins and gene markers for yeast and/or a bacterial host.The vector DNA is cleaved using an appropriate restriction endonuclease.The restricted DNA is ligated to the DNA fragment containing the yeastpromoter and to the DNA segment coding for the desulphatohirudin mutein.Prior to or after linking of the promoter and the mutant hirudin codingregion (or simultaneously as well), it is also possible to introducereplication origins and/or markers for yeast or a bacterial host. At allevents, the restriction and ligation conditions are to be chosen in sucha manner that there is no interference with the essential functions ofthe vector DNA and of the promoter. The hybrid vector may be built upsequentially or by ligating two DNA segments comprising all sequences ofinterest.

Various techniques may be used to join DNA segments in vitro. Blunt ends(fully base-paired DNA duplexes) produced by certain restrictionendonucleases may be directly ligated with T4 DNA ligase. More usually,DNA segments are linked through their single-stranded cohesive ends andcovalently closed by a DNA ligase, e.g. T4 DNA ligase. Suchsingle-stranded "cohesive termini" may be formed by cleaving DNA withanother class of endonucleases which produce staggered ends (the twostrands of the DNA duplex are cleaved at different points at a distanceof a few nucleotides). Single strands can also be formed by the additionof nucleotides to blunt ends or staggered ends using terminaltransferase ("homopolymeric tailing") or by simply chewing back onestrand of a blunt-ended DNA segment with a suitable exonuclease, such asλ exonuclease. A further approach to the production of staggered endsconsists in ligating to the blunt-ended DNA segment a chemicallysynthesized linker DNA which contains a recognition site for astaggered-end forming endonuclease and digesting the resulting DNA withthe respective endonuclease.

The components of the hybrid vector according to the invention, such asthe yeast promoter, structural gene for the mutant hirudin optionallyincluding a signal sequence, transcription terminator, the replicationsystem etc., are linked together in a predetermined order to assureproper function. The components are linked through common restrictionsites or by means of synthetic linker molecules to assure properorientation and order of the components.

A linear DNA vector is made by methods known in the art, e.g. linkingthe above yeast promoter to the mutant hirudin coding region in such amanner that the mutant hirudin coding region is controlled by saidpromoter, and providing the resulting DNA with a DNA segment containingtranscription termination signals derived from a yeast gene.

Joining of the DNA segments may be done as detailed above, viz. by bluntend ligation, through cohesive termini or through chemically synthesizedlinker DNAs.

In particular, in order to be efficiently expressed, the mutant hirudingene must be properly located with respect to sequences containingtranscriptional (yeast promoter) and translational functions (ribosomebinding sites). Firs fly, the ligation of the DNA segment comprising thepromoter with the mutant hirudin coding region has to be achieved in theproper orientation. If two orientations are possible the correct one isdetermined by conventional restriction analysis. Hybrid vectorscontaining an incorrectly oriented mutant hirudin gene insert arere-oriented by excising the gene insert with a suitable restrictionendonuclease and re-ligating the gene with the hybrid vector fragment.In any case improper orientation is avoided by ligating two DNA segmentseach with different restriction sites at their ends. Furthermore, theconstruction of the hybrid vector should be done in such a way that itallows correct transcription initiation and termination. As to thelatter point, the transcript should preferably end in a DNA sequencederived from yeast chromosomal DNA or yeast 2μ plasmid. Secondly, aproper reading frame must be established. Ordinarily, the nucleotidesequence of the promoter region and the mutant hirudin coding region isknown prior to ligation or can easily be determined so that there are noproblems in establishing the correct reading frame.

If the expression of the mature desulphatohirudin mutein foraccumulation in the cytoplasm is desired, signal sequences or partsthereof optionally following the promoter region and/or optionallypreceding the mature mutant hirudin coding region have to be eliminated,for example by digestion with an exonuclease, e.g. with Bal31. Apreferred region for directly joining a yeast promoter to the mutanthirudin coding sequence is between the major mRNA start site and the ATGof the gene naturally linked to the yeast promoter. For a junction inthis region the mutant hirudin coding sequence should have its own ATGfor translation initiation, or else it has to be provided with anadditional synthetic oligonucleotide. The yeast promoter may also belinked to the mutant hirudin coding sequence by means of a syntheticoligodeoxyribonucleotide as a connecting molecule. Thus, the promoterregion may be, if possible, restricted near its 3'-terminus so that itlacks a predetermined number of base pairs. Analogously, the mutanthirudin coding sequence may be restricted near its 5'-terminus. Asynthetic oligodeoxynucleotide can then be constructed in such a waythat, when joining the yeast promoter and the mutant hirudin codingsequence via the connecting oligodeoxynucleotide, the missing base pairsare restored including an ATG translation initiation signal and themutant hirudin coding sequence is in the proper reading frame relativeto the ATG initiation codon.

If only a desulphatohirudin fragment is used the production is dependenton the length of said fragment. Normally, heterologous proteins withless than 20 amino acids cannot be expressed and isolated in sufficientmounts from natural hosts like E. coli and S. cerevisiae. A possiblemethod for the expression of small proteins such as the carboxyterminalfragment of desulphatohirudin consists in fusing the small protein inquestion in frame to another protein thus creating a fusion protein.Several methods have been developed to liberate the desired protein fromits fusion protein (Nishikawa et al., Protein Engineering 1, 487-492(1987); Gardella et al., J. Biol. Chem. 265, 15854-15859, (1990); Altmanet al., Protein Engineering 4, 593-600 (1991)). Possible hosts are fungilike yeasts, preferably S. cerevisiae, or bacteria like E. coli.

Suitable endogenous fusion partners are highly expressed proteins.Preferred are proteins like eglin, β-galactosidase, β-lactamase, severalproteins involved in the tryptophan synthesis in E. coli, protein A orchloramphenicol acetyl transferase. In S. cerevisiae chimetic ubiquitinfusion proteins are preferably used (Sabin et al. Biotechnology 7,705-709 (1989).

The desired carboxyterminal fragment of desulphatohirudin has to beremoved from the fusion partner after expression and purification of thefusion protein. If there is no natural cleavage site between the desiredfragment and the fusion partner, this is generally done by means of alinker sequence linking the desired fragment to the fusion partner andcontaining a cleavage site which can selectively be cleaved by chemicalor enzymatic means. Such cleavage sites include, for example, amethionyl radical which is susceptible to the attack of cyanogenbromide, a polypeptide chain including the tetrapeptidyl radicalAsp-Asp-Asp-Lys which is cleaved by enterokinase after Lys or any othercleavage site susceptible to a specific cleavage with enzymes like V8protease, trypsin, peptidase yscα or yscF.

Another possibility for the production of short fragment is thesynthesis with a peptide synthesizer.

If the desulphatohirudin mutein or derivative is produced by a yeaststrain using a expression cassette including a signal sequence, theproduced desulphatohirudin is predominantly (i.e. more than 90%)secreted into the culture broth. The desulphatohirudin can be isolatedtherefrom by conventional means. For example, the first step consistsusually in separating the cells from the culture fluid by means ofcentrifugation. The resulting supernatant can be enriched fordesulphatohirudin by treatment with polyethyleneimine so as to removemost of the non-proteinaceous material, and precipitation of theproteins by saturating the solution with ammonium sulphate. Hostproteins, if present, can also be precipitated by means of acidificationwith acetic acid (for example 0.1%, pH 4-5). A further enrichment ofdesulphatohirudin can be achieved by extracting the acetic acidsupernatant with n-butanol. Other purification steps include, forexample, desalination, chromatographic processes, such as ion exchangechromatography, gel filtration chromatography, partition chromatography,HPLC, reversed phase HPLC and the like. The separation of theconstituents of the mixture is also effected by dialysis, according tocharge by means of gel electrophoresis or carrier-free electrophoresis,according to molecular size by means of a suitable Sephadex column, byaffinity chromatography, for example with antibodies, especiallymonoclonal antibodies, or with thrombin coupled to a suitable carrierfor affinity chromatography, or by other processes, especially thoseknown from the literature. In general, only a few purification steps arerequired in order to get a desulphatohirudin product which isessentially free of contaminants.

The test with anti-desulphatohirudin or anti-desulphatohirudinantibodies (for example monoclonal antibodies), the thrombin test (M. U.Bergmeyer (ed.), Methods in Enzymatic Analysis 1983, Vol. II, p.314-316, Verlag Chemie, Weinheim (FRG)) or the blood coagulation test(F. Markwardt et al., Thromb. Haemost. (1982), 47, 226) can be used todetect the desulphatohirudin activity during the isolation andpurification steps. It is also possible to use chromatographical means,such as HPLC.

It is therefore an object of the invention to provide an expressioncassette for a desulphatohirudin according to the invention, consistingof a promoter operably linked to a first DNA sequence encoding a signalpeptide linked in the proper reading frame to a second DNA sequencecoding for said desulphatohirudin mutein or derivative, and a DNAsequence with the transcription termination signals.

It is a further object of the invention to provide a method for theproduction of such desulphatohirudin conjugates consisting essentiallyof two to four desulphatohirudin muteins or derivatives, consisting of

production of a suitable desulphatohirudin monomer e.g. by expression ofa suitable desulphatohirudin in a suitable host,

isolation of the desulphatohirudin,

protection of the reacitve amino acid side chains if necessary,

linking of the desulphatohirudin monomer to a suitable linker ordirectly linking of two desulphatohirudin monomeres together e.g. via adisulfide bond.

Pharmaceutical compositions

The novel desulphatohirudin conjugates obtainable in accordance with thepresent invention have valuable pharmacological properties and can, likemonomeric desulphatohirudin extracted from leeches, be usedprophylactically or, especially, therapeutically.

The desulphatohirudin conjugates according to the invention are, likenatural hirudin, potent inhibitors of thrombin. They have, for example,a K_(i) value of 10⁻⁹ M to 10⁻¹³ M. The desulphatohirudin conjugates arecompletely specific to thrombin and exhibit no interactions with otherproteinases of the blood coagulation system. The toxicity is extremelylow. Similarly, no hypersensitivity reactions or allergic reactions areobserved. Because of the increased plasma half life of thedesulphatohirudin conjugates, the administration of the dally dose ofdesulphatohirudin in a single portion is possible.

The novel desulphatohirudin conjugates according to the invention cantherefore be used analogously to natural hirudin for the therapy andprophylaxis of thromboses and thromboembolisms, including theprophylaxis of post-operative thromboses, for acute shock therapy (forexample for septic or polytraumatic shock), for the therapy ofconsumption coagulopathies, in haemodialyses, haemoseparations and inextracorporeal blood circulation.

The invention relates also to pharmaceutical compositions that contain atherapeutically effective amount of at least one of the compoundsaccording to the invention or a pharmaceutically acceptable saltthereof, optionally together with a pharmaceutically acceptable carrierand/or adjuncts.

These compositions can be used especially in the above-mentionedindications, when they are administered, for example, parenterally, suchas intravenously, intracutaneously, subcutaneously or intramuscularly.

The invention relates also to the use of the novel compounds accordingto the invention and to pharmaceutical compositions containing them forthe prophylactic and therapeutic treatment of the human or animal body,especially for the above-mentioned clinical syndromes, especially forinhibiting the coagulation of blood inside and outside the human oranimal body.

The dosage depends especially on the specific form of administration andon the purpose of the therapy or prophylaxis. The size of the individualdoses and the administration regime can best be determined by way of anindividual judgement of the particular case of illness; the methods ofdetermining relevant blood factors required for this purpose arefamiliar to the person skilled in the art. Normally, in the case of aninjection the therapeutically effective amount of the compoundsaccording to the invention is in a dosage range of from approximately0.005 to approximately 0.1 mg/kg body weight. A range of fromapproximately 0.01 to approximately 0.05 mg/kg body weight is preferred.The administration is effected by intravenous, intramuscular orsubcutaneous injection. Accordingly, pharmaceutical compositions forparenteral administration in single dose form contain per dose,depending on the mode of administration, from approximately 0.4 toapproximately 7.5 mg of the compound according to the invention. Inaddition to the active ingredient these pharmaceutical compositionsusually also contain a buffer, for example a phosphate buffer, which isintended to keep the pH value between approximately 3.5 and 7, and alsosodium chloride, mannitol or sorbitol for adjusting the isotonicity.They may be in freeze-dried or dissolved form, it being possible forsolutions advantageously to contain an antibacterially activepreservative, for example from 0.2 to 0.3% 4-hydroxybenzoic acid methylester or ethyl ester.

Another embodiment of the invention concerns an aqueous formulationcomprising water, at least one of the compounds according to theinvention and calcium, magnesium or zinc ions in the form of a waterinsoluble salt.

The water insoluble salt is preferably a phosphate as they are veryinsoluble.

This formulation may have a pH of from 4 to 11, preferably from 5 to 9.The concentration of the metal salt may be from 100 mM to 600 mM,preferably from 100 mM to 300 mM and most preferably about 150 mM. Ifthe concentration is higher than a usable physiological concentration,the formulation may be diluted to physiological concentration beforeuse. The concentration of the compounds according to the invention maybe from 1 to 600 mg/ml, preferably from 20 to 80 mg/ml. If a highconcentration is present it may be diluted, e.g. to 20 to 80 mg/ml rangebefore use.

The particle size of the water insoluble salt may be from 10 to 30 μmdiameter, preferably from 10 to 20 μm.

This formulation may be prepared by precipitating the water insolublesalt in situ in an aqueous solution of the compounds according to theinvention. For example the chloride of the chosen metal (Ca, Mg or Zn)may be mixed with alkali metal phosphate. The pH of the resultingformulation may then be adjusted using e.g. hydrochloric acid or sodiumhydroxide as appropriate. The preferred metal for this formultation iszinc.

The formulations of the invention may also contain a sugar such assucrose, trehalose or, preferably, mannitol.

In addition to the compositions described above that are intended fordirect medicinal use in the body of a human or an animal, the presentinvention relates also to pharmaceutical compositions for medicinal useoutside the living body of humans or animals. Such compositions are usedespecially as anticoagulant additives to blood that is being subjectedto circulation or treatment outside the body (for example extracorporealcirculation or dialysis in artificial kidneys), preservation ormodification (for example haemoseparation). Such compositions, such asstock solutions or alternatively compositions in single dose form, aresimilar in composition to the injection compositions described above;however, the amount or concentration of active ingredient isadvantageously based on the volume of blood to be treated or, moreprecisely, on its thrombin content. In this connection it must be bornein mind that the active ingredients according to the invention (in freeform) completely deactivate approximately 5 times the amount by weightof thrombin, are physiologically harmless even in relatively largeamounts, and are eliminated from the circulating blood rapidly even inhigh concentrations so that there is no risk of overdose, even, forexample, during transfusions. Depending on the specific purpose, thesuitable dose is from approximately 0.01 to approximately 1.0 mg of theactive ingredient/liter of blood, although the upper limit may still beexceeded without risk.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following experimental part various embodiments of the presentinvention are described with reference to the accompanying drawings inwhich:

FIG. 1 is a schematic illustration of the refolding of the Cys³³HV1--Cys³³ HV1 dimmers.

EXPERIMENTAL PROCEDURES

All DNA manipulations are--if not otherwise noted--carried out accordingto standard protocols (e.g. Maniatis, T. et al.: Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

EXAMPLE 1: Construction of plasmid pDP34/GAPFL-YHIR (CYS33):

The expression in yeast (Saccharomyces cerevisiae) ofdesulphatohirudin-Cys³³, a mutant of the recombinant desulphatohirudinderivative 1 (HV1) (J. Dodt et al., FEBS Lett. 165 (1984), 180-184) withcysteine replacing aspartic acid at amino acid position 33, is achievedby changing the nucleotide sequence coding for desulphatohirudin. Themethod of Polymerase Chain Reaction (PCR) ("PCR Protocols: A guide tomethods and applications" M. A. Innis, D. H. Gelfand, J. J. Sninsky, T.J. White (eds.). Academic Press, N.Y., 1990) is used to replace GAC withTGT in the expression cassette for the mutant desulphatohirudin. TGTcodes for cysteine and therefore leads to the expression ofdesulphatohirudin-Cys³³ (Cys³³ HV1).

The following PCR primers are used:

    __________________________________________________________________________    (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4)                                                   complementary to nucleotide                                                   positions of template DNA                           __________________________________________________________________________    (1)                                                                             5'-TCGGTCGACGCTCTCCCTTATGCG-3'                                                                        7492-7494, 1-21                                     (2)                                                                             5'-TTGGGTTCTTGTGGTGAAAAGAACCAATGTG-3'                                                                  626-656                                            (3)                                                                             5'-CTTTTCACCACAAGAACCCAAGATACACTTG-3'                                                                  646-616                                            (4)                                                                             5'-GCCAAGCTTGGCTGCAGATTTTAATC-3'                                                                      1122-1100                                           __________________________________________________________________________

The oligodeoxynucleotide primers are complementary to nucleotidesequences (see above) in the template DNA pJDB207/GAPFL-YHIR (EP-A-340170) SEQ ID NO: 5. Primers (1) and (4) are the upstream and downstreamprimers for amplification of the template DNA; primers (2) and (3) arethe internal primers which are partly complementary to each other andboth cover the site of mutation (underlined). The PCR primers (1) and(3) and primers (2) and (4) are used pairwise in separate reactions toamplify parts of the template DNA and to introduce the mutation. BothPCR products overlap in the region of the mutation. In a second reactionboth products are mixed and amplified with primers (1) and (4).

0.1 mg of template DNA of plasmid pJDB207/GAPFL-YHIR (EP-A-340 170) aremixed with 1 mg each of the primers (1) and (3) and alternatively withprimers (2) and (4) for amplification of the corresponding DNA fragmentsaccording to standard PCR protocols (see above) using 30 cycles with 1min. for DNA synthesis at 72° C., 10 sec. for DNA strand separation at93° C. and 20 sec. for annealing at 50° C. The DNA fragments arepurified by phenol extraction and precipitated in ethanol.

The second overlap-extension reaction contains 0.1 mg each of the PCRproducts amplified in the first reaction (see above) and 1 mg each ofthe PCR primers (1) and (4). 30 cycles with 1,5 min. for DNA synthesisat 72° C., 20 sec. for strand separation at 93° C. and 40 sec. forannealing at 50° C. are used.

The amplified DNA fragment is restricted with SalI and HindIII. The 1.1kb SalI-HindIII fragment is cloned into SalI, HindIII cut vector pUC9(Sigma®). Several clones are checked by restriction analysis and theinsert of one correct clone is sequenced. The coding sequence of thisclone contains the TGT codon coding for Cys³³.

The 1.1 kb SalI-HindIII insert is the mutant desulphatohirudinexpression cassette comprising the 276 bp SalI-BamHI fragment of pBR322,a BamHI/BgIII junction and the 194 bp GAPFL promoter with an EcoRIlinker at position -5. The EcoRI site joins the promoter to the codingsequence of the PHO5 signal sequence and [Cys³³ ]HV1. The 3'terminalBamHI site is followed by the 377 bp PHO5 transcription terminator and aHindIII site.

The 1.1 kb expression cassette is cloned as a SalI-blunt end fragmentinto high copy number yeast expression vector pDP34 (DSM 4473) cut withBamHI (converted to blunt end by Klenow polymerase) and SalI. Theresulting plasmid is referred to as pDP34/GAPFL-YHIR (CYS33). Thesequences of the Cys³³ HV1 part and the corresponding protein are givenin SEQ ID NO: 7 and SEQ ID NO: 8.

EXAMPLE 2: Yeast transformation and fermentation

Saccharomyces cerevisiae strain TR1456 is constructed as disclosed inEP-A-341 215. Starting with Saccharomyces cerevisiae strain H449 (DSM4413), in two subsequent series of experiments the two carboxypeptidasesyscα and yscY are removed from strain H449 by disruption of theirencoding genes KEX1 and PRC1, respectively. First, the gene encodingyscα, KEX1, is disrupted. For this purpose, strain H449 is transformedwith a DNA fragment encoding the KEX1 gene, with the full URA3 geneinserted in the middle of the KEX1 coding region. Uracil prototrophictransformants are selected and tested for the absence of yscα activity.Next, the URA3 gene inserted at the KEX1 locus is disrupted bytransformation with a plasmid containing a disrupted version of the URA3gene, ura3Δ5 (see EP-A-341 215). Transformants which are uracilauxotrophic are selected and in the following step disrupted in theirendogenous PRC1 gene coding for the carboxypeptidase yscY. Theexperiment is carried out in a totally analogous manner as described forthe disruption of KEX1. The finally resulting isogenic derivative ofstrain H449 is called TR1456 and has the following genotype:

    MATa, prb1, cps1, prc1::ura3, kex1::ura3, leu2-3,112, ura3Δ5, [cir.sup.0 ].

Yeast strain TR 1456 is transformed with plasmid pDP34/GAPFL-YHIR(CYS33)using the transformation protocol of Hinnen et al. (Proc. Natl. Acad.Sci. USA, 75 (1978) 1929-1933). Transformed yeast cells are grown underuracil selection. A single transformed yeast colony is referred to as

    Saccharomyces cerevisiae TR 1456 /pDP34/GAPFL-YHIR(CYS33).

The transformant is fermented for two successive precultures of 10 mlminimal medium lacking uracil, for the actual production run in complexmedium without selection. Crude [Cys³³ ]HV1 is isolated from the culturebroth as described in EP-A-340 170.

EXAMPLE 3: Preparation, unfolding and refolding of Cys³³ HV1

[Cys³³ ]HV1 is expressed in yeast and excreted into the medium asdescribed for native hirudin (Meyhack et al. (1987), Thromb. Res.Suppl., VII, 33). Cells are removed by centrifugation and proteinsshowing antithrombin activity are removed from supernatant by adsorbtionon XAD-7 resin. A mixture of active proteins is eluted with ammoniumacetate buffer (0.2M, ph 8.5) and applied to a Q-sepharose fast-flowion-exchange column. This column is eluted with ammonium formiate buffer(25 mM), using gradient elution from 0% A (buffer adjusted to pH 5.0with formic acid) to 85% B (buffer adjusted to pH 3.0 with formic acidand containing 0.5M sodium chloride) within 50 min. Fractions showingantithrombin activity are pooled and lyophilized to dryness.

The crude [Cys³³ ]HV1 (4 mg) is reduced for 2 h at 23° C. in 0.75 ml ofTris-HCl buffer (0.5M, pH 8.4) containing 5M guanidine hydrochloride(GdmCl) and 0.1M dithiothreitol. The sample is passed through a PD-10column (Pharmacia) equilibrated in 50 mM sodium bicarbonate buffer, pH8.3. Reduced/denatured sample is collected in 1.2 ml and immediatelydiluted with the same sodium bicarbonate buffer to a final concentrationof 1 mg/ml (140 mM). The folding is carried out at 23° C. and foldingintermediates are trapped in a time course manner by acidifying aliquotsof folding sample with 2 volumes of 4% trifluoroacetic acid.

EXAMPLE 4: HPLC characterization of the folding intermediates

[Cys³³ ]HV1 is refolded in 50 mM NaHCO₃ (pH 8.3) at 23° C. (see FIG. 1).The folding intermediates are trapped by acid in a time course mannerand analyzed by HPLC using the following conditions:

Column: Vydac C-18 for peptides and proteins, 5 mm;

Solvent A: water containing 0.1% trifluoroacetic acid;

Solvent B: acetonitrile/water (9:1, v/v) containing 0.1% trifluoroaceticacid.

Gradient: 24% to 43% solvent B linear in 20 min.;

Detection: at 214 nm.

The reduced/denatured (R) Cys³³ HV1 is eluted at 17.7 min.

"NM" is active Cys³³ HV1 monomer (13.5 min.).

"ND" is Cys³³ HV1 dimer.

Inset in FIG. 1 is crude [Cys³³ ]HV1 analyzed by the same HPLCconditions.

The heterogeneity of the crude product arises from the improper foldingafter biosynthesis or sample destruction during the purification.

Active [Cys³³ ]HV1 monomer (NM) is formed first (FIG. 1) with a firstorder rate constant of 0.05±0.01 min.⁻¹ under the conditions described.Oxidation of Cys³³ between two active [Cys³³ ]HV1 monomers subsequentlyoccurred and the dimer is generated. The folding is highly efficient andthe yield is greater than 85-90%. At pH higher than 8.3, the foldingproceeds at an even faster speed.

EXAMPLE 5: Characterization of the [Cys³³ ] desulphatohirudin monomerand dimer

The amino acid composition is determined by the dimethylaminoazobenzenesulfonyl (dabsyl) chloride method (Chang et al. Anal. Biochem. (1991),197, 52-58.), which allows direct quantization of the disulfide content.The amino acid sequence was analyzed by an Applied Biosystem Inc. 470Aprotein sequencer. The antithrombin activity of desulphatohirudinderivatives was assessed by their ability to inhibit α-thrombin fromdigesting Chromozym TH (Boehringer Mannheim), using the describedprotocol (Stone et al. Biochemistry (1986), 25, 4622-4628; Chatrenet etal. J. Biol. Chem. (1992), 267, 3038-3043). The molecular weight ofdesulphatohirudin is 13916, determined by the laser desorptionionization mass spectrometry using a home-built instrument (Boernsen etal. Chimia (1990), 44, 412-416).

The [Cys³³ ]HV1--[Cys³³ ]HV1 dimer contains no free cysteine asconfirmed by amino acid analysis.

The measurement of thrombin inhibition is performed at the concentrationof 1 nM of desulphatohirudin and 1.5 nM of α-thrombin, the dimerexhibits, at equal weight basis, essentially the same inhibitoryactivity as wild type desulphatohirudin (82±5%).

EXAMPLE 6: The dimer and monomer are inter-convertible

Unlike the three intra disulfides of desulphatohirudin, the Cys³³--Cys³³ bond of the dimer is not stabilized by non-covalentinteractions. It is therefore possible to selectively cleave the Cys³³--Cys³³ bond under mild reducing conditions without disrupting the threedisulfides located within the active core domain. Experiments arecarried out in order to find out an optimum condition which allowsquantitative conversion of the dimer to the monomer. This can beachieved by using 0.5 to 2.5 mM of β-mercaptoethanol at roomtemperature. At higher concentration of β-mercaptoethanol, the monomerrapidly disintegrates as a consequence of the reduction of Cys⁶ -Cys¹⁴,Cys¹⁶ -Cys²⁸ and Cys²² -Cys³⁹ disulfide bonds.

EXAMPLE 7: Pharmaceutical composition containing a desulphatohirudindimer for parenteral administration

The lyophilized [Cys³³ ]HV1-[Cys³³ ]HV1 dimer according to example 3 isdissolved in 0.9% NaCl solution to a final concentration of 0.2 mg/ml or2 mg/ml. These solutions are sterilized by ultrafiltration (membraneswith 0.22 μm pores).

The sterilized solutions can be used directly, for example, forintravenous administration.

EXAMPLE 8: Effect of subcutaneously administered Hirudin HV1 and [Cys³³]HV1-[Cys³³ ]HV1 dimer on ex vivo plasma APTT in the rat

(APTT=activated partial thrombin time)

[Cys³³ ]HV1--[Cys³³ ]HV1 dimer is dissolved in saline to a concentrationof 4.5 mg/ml and unmodified hirudin-monomer is dissolved in saline to aconcentration of 3.0 mg/ml (these doses are chosen based on the in vitroresults in order to achieve a similar effect on APTT at 1 h). Solutionsare administered subcutaneously to rats (1 ml/kg). At various timesafter administration, blood (2 ml) is collected by cardiac puncturedirectly into one tenth volume trisodium titrate solution (3.8% w/v) andimmediately mixed. The anticoagulated blood is centrifuged at 2500 g for20 min to obtain platelet free plasma.

Plasma samples are assayed for APTT using standard methods on theInstrumentation Laboratory ACL 300R coagulometer. The method for APTTdetermination utilizes the Instrumentation Laboratory ellagic acid testkit--plasma (50 μl) is incubated with ellagic acid activating reagent(50 μl) at 37° C. for 5 min prior to the addition of calcium chloridesolution (50 μl of 25 mM solution) and the time taken for the plasmaclot is recorded automatically, the end point being determined opticallyby the light scattered when the fibrin network formed (see table 1).

                  TABLE 1                                                         ______________________________________                                        Time                                                                          (hours)   HV1       [Cys.sup.33 ]-[Cys.sup.33 ]HV1 dimer                      ______________________________________                                        0         1.00 ± 0.02                                                                          1.00 ± 0.02                                            1         2.44 ± 0.07                                                                          2.29 ± 0.08                                            4         1.43 ± 0.05                                                                          2.07 ± 0.25                                            6         0.95 ± 0.02                                                                          1.44 ± 0.02                                            ______________________________________                                    

The results are expressed as a multiple of the means control APTT valueand presented as mean±standard error of the mean (n=5).

EXAMPLE 9: Pharmaceutical composition

Solution A containes 2.43M ZnCl₂, and 0.455M Na₂ HPO₄ in water at pH2.5, pH adjusted with HCl. Solution B consists of 0.6M NaOH, 0.25M NaCl.A solution C is made by mixing 2 parts of a stock solution of [Cys³³]HV1--[Cys³³ ]HV1 dimer in water (80 mg/ml) with 1.05 parts of solutionA (ratio 2:1.05, v/v ). The formulation is obtained by mixing water,solution B, and solution C in the weight ratio of 0.66:0.1883:0.4,respectively.

Similar mixtures are made using CaCl₂ or MgCl₂ instead of ZnCl₂.

EXAMPLE 10: Pharmaceutical composition

Solution A1 containes 165 mM ZnCl₂, 2.11 mM Na₂ HPO₄ and 37.8 mM HCl inwater. Solution B1 contains 0.6N NaOH. [Cys³³ ]HV1-[Cys³³ ]HV1 dimer(powder) is added to 31.7 parts by volume solution A1, followed by 55parts by volume mannitol solution (198 mM) and then 13.3 parts by volumesolution B1. The solution becomes turbid as the depot is formed. [Cys³³]HV1--[Cys³³ ]HV1 dimer is used in an amount of 20 mg/ml water used. NopH adjustment is needed.

EXAMPLE 11: Determination of the kinetic parameters for inhibition ofhuman thrombin by the [Cys³³ ]HV1--[Cys³³ ]HV1 dimer

The concentration of the hirudin is determined by titration against 2.0nM thrombin in presence of 200 μM D-Val-Leu-Arg-p-nitroanilide asdescribed by Wallace et al., Biochemistry (1989), 28, 10079-10084.

Based on weight and a molecular mass of 7000 for one subunit, thereaction of active molecules is 0.97. This finding demonstrated thatboth molecules in the dimer are active.

The kinetic parameters of the [Cys³³ ]HV1--[Cys³³ ]HV1 dimer aredetermined from progress-curve experiments as described by Stone et al.Biochemistry (1986), 25, 4622-4628 and Braun et al. Biochemistry,(1988), 27, 6517-6522. For each determination, the formation ofp-nitroaniline from D-Phe-pipecolyl-Arg-p-nitroanilide is monitored insix assays, one in absence of inhibitor and the others with differentconcentrations of inhibitor ranging from 10 to 60 pM for the [Cys³³]HV1--[Cys³³ ]HV1 dimer. The substrat concentration is 100 μM and theenzyme concentration is 20 pM.

The kinetic parameters are determined twice and values given in table 2represent the weightened means of the estimates.

                  TABLE 2                                                         ______________________________________                                                       Parameter                                                                       10.sup.8 k.sub.1                                                                         K.sub.1                                           Inhibitor        (M.sup.-1 s.sup.-1)                                                                      (pM)                                              ______________________________________                                        [Cys.sup.33 ]HV1-[Cys.sup.33 ]HV1 dimer                                                        (0.51 ± 0.03)                                                                         0.330 ± 0.038                                  HV1              (1.37 ± 0.03)                                                                         0.237 ± 0.006                                  ______________________________________                                    

Deposition of microorganisms

The following microorganism strains were deposited at the DeutscheSammlung von Mikroorganismen (DSM), Mascheroder Weg 1b, D-3300Braunschweig (deposition dates and accession numbers given):

Saccharomyces cerevisiae H449: Feb. 18, 1988, DSM 4413;

Escherichia coli JM109/pDP34: Mar. 14, 1988, DSM 4473;

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: /product="Synthetical PCR Primer"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      TCGGTCGACGCTCTCCCTTATGCG24                                                    (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..31                                                           (D) OTHER INFORMATION: /product="Synthetical PCR Primer"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      TTGGGTTCTTGTGGTGAAAAGAACCAATGTG31                                             (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..31                                                           (D) OTHER INFORMATION: /product="Synthetical PCR Primer"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTTTTCACCACAAGAACCCAAGATACACTTG31                                             (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..26                                                           (D) OTHER INFORMATION: /product="Synthetical PCR Primer"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GCCAAGCTTGGCTGCAGATTTTAATC26                                                  (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1130 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_signal                                                     (B) LOCATION: 1..1130                                                         (D) OTHER INFORMATION: /function="first 1130 nucleotides                      of pJDB207/GAPFL-YHIR (EP-A-340 170)"                                         (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..276                                                          (D) OTHER INFORMATION: /product="SalI - BamHI fragment of                     pBR322"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 277..284                                                        (D) OTHER INFORMATION: /function="BamHI/BglII linker"                         (ix) FEATURE:                                                                 (A) NAME/KEY: promoter                                                        (B) LOCATION: 285..478                                                        (D) OTHER INFORMATION: /function="GAPFL promoter"                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 479..484                                                        (D) OTHER INFORMATION: /product="EcoRI linker"                                (ix) FEATURE:                                                                 (A) NAME/KEY: misc_signal                                                     (B) LOCATION: 488..538                                                        (D) OTHER INFORMATION: /function="PHO5 signal sequence"                       (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 539..733                                                        (D) OTHER INFORMATION: /product="Hirudin HV1"                                 /standard_name="HV1"                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: terminator                                                      (B) LOCATION: 737..1113                                                       (D) OTHER INFORMATION: /standard_name="PHO5 terminator"                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1117..1122                                                      (D) OTHER INFORMATION: /function="M13 cloning site"                           (ix) FEATURE:                                                                 (A) NAME/KEY: misc_signal                                                     (B) LOCATION: 734..736                                                        (D) OTHER INFORMATION: /function="Translation terminator"                     /standard_name="TAG"                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGC60                CGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCC120               CGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGC180               GAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGG240               CGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCTCCCTGAAAAAAAAGGTTG300               AAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTA360               TTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGT420               CTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAATAAAGA480               ATTCAAAATGTTTAAATCTGTTGTTTATTCAATTTTAGCCGCTTCTTTGGCCAATGCAGT540               TGTTTACACCGACTGTACCGAATCTGGTCAAAACTTGTGTTTGTGTGAAGGTTCTAACGT600               TTGTGGTCAAGGTAACAAGTGTATCTTGGGTTCTGACGGTGAAAAGAACCAATGTGTTAC660               CGGTGAAGGTACCCCAAAGCCACAATCTCACAACGACGGTGACTTCGAAGAAATCCCAGA720               AGAATACTTGCAATAGGATCCTGGTACGTTCCTCAAGGTGCTCGTGTCTACACCGAAAAA780               TTCCAATGTTCTAACGACACCTACGTCAGATACGTCATTAACGATGCTGTTGTTCCAATT840               GAAACCTGTTCCACTGGTCCAGGGTTCTCTTGTGAAATCAATGACTTCTACGACTATGCT900               GAAAAGAGAGTAGCCGGTACTGACTTCCTAAAGGTCTGTAACGTCAGCAGCGTCAGTAAC960               TCTACTGAATTGACCTTCTACTGGGACTGGAACACTACTCATTACAACGCCAGTCTATTG1020              AGACAATAGTTTTGTATAACTAAATAATATTGGAAACTAAATACGAATACCCAAATTTTT1080              TATCTAAATTTTGCCGAAAGATTAAAATCTGCAGCCAAGCTTTGAAGAAA1130                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..65                                                           (D) OTHER INFORMATION: /note="hirudin variant 1 (HV1)"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        AspGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              505560                                                                        Gln                                                                           65                                                                            (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 1..195                                                          (D) OTHER INFORMATION: /product="[33-Cys]HV1"                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GTTGTTTACACCGACTGTACCGAATCTGGTCAAAACTTGTGTTTGTGTGAAGGTTCTAAC60                GTTTGTGGTCAAGGTAACAAGTGTATCTTGGGTTCTTGTGGTGAAAAGAACCAATGTGTT120               ACCGGTGAAGGTACCCCAAAGCCACAATCTCACAACGACGGTGACTTCGAAGAAATCCCA180               GAAGAATACTTGCAA195                                                            (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..65                                                           (D) OTHER INFORMATION: /note="[33-Cys]HV1"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      ValValTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys                              151015                                                                        GluGlySerAsnValCysGlyGlnGlyAsnLysCysIleLeuGlySer                              202530                                                                        CysGlyGluLysAsnGlnCysValThrGlyGluGlyThrProLysPro                              354045                                                                        GlnSerHisAsnAspGlyAspPheGluGluIleProGluGluTyrLeu                              505560                                                                        Gln                                                                           65                                                                            __________________________________________________________________________

We claim:
 1. A conjugate consisting of two to four residues ofdesulphatohirudin muteins or derivatives having hirudin activity whereinthe conjugate is not a fusion protein, the residues of desulphatohirudinmuteins or derivatives having hirudin activity are not connected viaglutaraldehyde or carbodiimide, and wherein said residues are linkedwithin the N-terminal domain of desulphatohirudin.
 2. A conjugateaccording to claim 1, consisting of two residues of desulphatohirudinmuteins or derivatives having hirudin activity.
 3. A conjugate accordingto claim 1, consisting of two residues of desulphatohirudin muteins orderivatives, of HV1, HV2, or HV3 (PA), having hirudin activity.
 4. Aconjugate according to claim 1, consisting of two residues ofdesulphatohirudin HV1 muteins or derivatives having hirudin activity. 5.A conjugate according to claim 1, wherein the desulphatohirudins ordesulphatohirudin derivatives are connected directly or via a linkergroup.
 6. A conjugate according to claim 1, wherein two cysteines in thedesulphatohirudin muteins or derivatives having hirudin activity areconnected directly or via a linker group.
 7. A conjugate according toclaim 1, wherein the desulphatohirudin muteins or derivatives areconnected directly via a disulfide bond.
 8. A conjugate according toclaim 1, wherein the monomers of the two desulphatohirudin muteins orderivatives, or the monomer of desulphatohirudin mutein or derivativeand the linker group, are linked in the region between amino acidresidues 7 to
 50. 9. A conjugate according to claim 8, wherein themonomers of the two desulphatohirudin muteins or derivatives, or themonomer of desulphatohirudin mutein or derivative and the linker group,are linked in the region between amino acid residues 30 to
 40. 10. Aconjugate according to claim 1, comprising desulphatohirudins whereinone or more amino acids are replaced by one or more radicals capable ofbeing crosslinked.
 11. A conjugate according to claim 10, comprisingdesulphatohirudins wherein one amino acid of desulphatohirudin isreplaced by an amino acid selected from the group consisting of asparticacid, glutamic acid, lysine and cysteine.
 12. A conjugate according toclaim 11, comprising desulphatohirudins wherein one amino acid isreplaced by cysteine.
 13. A conjugate according to claim 12, comprisingtwo desulphatohirudin HV1 wherein Asp³³ is replaced by Cys.
 14. Adesulphatohirudin HV1, wherein Asp³³ is replaced by Cys (SEQ ID NO: 8).15. A first desulphatohirudin mutein or derivative, having hirudinactivity, being capable of being crosslinked to a seconddesulphatohirudin mutein or derivative also having hirudin activity,wherein said second desulphatohirudin mutein or derivative is either thesame as or different from the first desulphatohirudin mutein orderivative.
 16. A method for preparing a pharmaceutical compositioncomprising combining the compound of claim 1 with conventional carriers.17. A pharmaceutical composition comprising a conjugate consisting oftwo to four residues of desulphatohirudin muteins or derivativesaccording to claim 1, or which comprises said compounds together withconventional auxiliaries, typically carriers and diluents.
 18. Anaqueous formulation comprising water, a compound according to claim 1and calcium, magnesium or zinc ions to form a water insoluble salt. 19.A formulation according to claim 18, wherein the water insoluble salt isa phosphate.
 20. A formulation according to claim 18, wherein theconcentration of the metal salt is from 100 mM to 600 mM.
 21. Aformulation according to claim 18, wherein the concentration of thecompound according to claim 1 is from 1 to 600 mg/ml.
 22. A formulationaccording to claim 18, wherein the particle size of the water insolublesalt is from 10 to 30 μm diameter.
 23. A pharmaceutical compositioncomprising a therapeutically effective amount of at least one of theconjugates according to claim 1 or a pharmaceutically acceptable saltthereof for use in a method for the therapy and prophylaxis ofthromboses and embolism.
 24. An expression vector comprising anexpression cassette for a desulphatohirudin according to claim 5,consisting of a promoter operably linked to a first DNA sequenceencoding a signal peptide linked in the proper reading frame to a secondDNA sequence coding for said desulphatohirudin mutein or derivative, anda DNA sequence with the transcription termination signals.
 25. Anexpression cassette according to claim 24 wherein the promoter is theGAPFL or the CUP1 promoter.
 26. An expression cassette according toclaim 24 wherein the signal sequence is the PHO5 signal sequence.
 27. Anexpression cassette according to claim 24 wherein the desulphatohirudinmutein or derivative is [Cys³³ ]HV1 (SEQ ID NO: 7).
 28. An expressioncassette according to claim 24 wherein the terminator is the PHO5terminator.
 29. Process for the production of a conjugate according toclaim 1, consisting ofexpression of a suitable desulphatohirudin in asuitable host, isolation of the desulphatohirudin, protection of thereactive amino acid side chains if necessary, linking of thedesulphatohirudins to a suitable linker or directly linking of twodesulphatohirudins together.
 30. A process according to claim 29 whereinthe desulphatohirudins are linked via a disulfide bond.
 31. A processaccording to claim 30, wherein the desulphatohirudins are unfolded andrefolded after isolation.
 32. A method for denaturing and renaturingdesulphatohirudin to a biologically active polymeric or dimeric formcomprising the steps:denaturing desulphatohirudin as defined above withchaotropic agent, renaturing of the desulphatohirudin under refoldingconditions, and linking of the desulphatohirudins by means of a linkeror directly.
 33. A method for the treatment of a human or animal in needof thrombin inhibition, said method comprising administering to thehuman or animal an effective amount of the conjugate of claim 1 in apharmaceutically acceptable carrier.